This application addresses Challenge Area 06, Enabling technologies;Specific challenge topic 06-MH-103, "New technologies for neuroscience research" MicroRNAs are believed to contribute to schizophrenia, autism, Tourette's syndrome, mood disorders, and fragile X syndrome. MicroRNA networks in brain are quite complex, however, largely because each microRNA can have hundreds of mRNA targets and each target can, potentially, be regulated by dozens of microRNAs. Research in this area has hit a roadblock because of two major gaps in the understanding of microRNA function. First, what determines which microRNAs bind to a specific mRNA target is very poorly understood. This problem has not been approachable because there has not been an empirical way to determine which particular microRNA interactions occur under specific conditions. Second, the populations of mRNA targets regulated by individual microRNAs are poorly defined. Linking the dysregulation of a microRNA pathway to a particular mRNA target requires this information, which cannot be obtained through bioinformatics alone. We have developed a general method to solve the first problem and will apply it to identify microRNAs that interact with the 3'UTR sequences of mRNAs encoding p250GAP and LimKI, two proteins that have been linked to dendritic spine formation and synaptic plasticity. We have also optimized a complementary method for identifying additional targets of the two microRNAs, miR132 and miR134, that have been proposed to regulate p250GAP and LimKI expression at the synapse. The novel methods outlined in this proposal will have a major impact on the understanding of how microRNAs contribute to synaptic plasticity that could lead to new therapeutic approaches. The approach that we have developed utilizes a fusion gene containing a 3'UTR probe linked to the sequence, MS2, which is recognized by the bacteriophage MS2 binding protein. The MS2-tagged 3'UTR probes will be introduced into primary cortical cells and transgenic mice, and complexes containing the associated microRNAs will be purified by affinity chromatography and identified by multiplex PCR. We have already established the efficacy of this approach and will now characterize the populations of microRNAs interacting with the p250GAP and LimKI transcripts and determine whether these associations are influenced by growth factors and neuronal activity using cell culture and transgenic mouse models. We will then identify new mRNA targets of miR132 and miR134 by immunopurifying RNA-induced silencing complexes (RISC) containing an epitope-tagged version of Ago2. A few targets of these microRNAs have been identified using various prediction algorithms, but most remain to be discovered. These studies will allow us to characterize the full complement of miR132 and miR134 targets and determine whether miR132 and miR134 regulate distinct, overlapping, or functionally related sets of mRNA targets. We predict that the majority of miR132 and miR134 targets will be distinct, but that these targets will converge on pathways involved in mediating synaptic plasticity. PUBLIC HEALTH RELEVANCE: This project describes a novel method for determining which microRNAs bind to the 3'UTR sequences of p250GAP and LimKI, two proteins that have been proposed to regulate the formation of synaptic spines in response to growth factors and neuronal activity. This method is critical for understanding the contribution of microRNA pathways to neurological and psychiatric disease.